Ciliate telomerase RNA loop IV nucleotides promote hierarchical RNP assembly and holoenzyme stability

Abstract

Telomerase adds simple-sequence repeats to chromosome 39 ends to compensate for the loss of repeats with each round of genome replication. To accomplish this de novo DNA synthesis, telomerase uses a template within its integral RNA component. In addition to providing the template, the telomerase RNA subunit (TER) also harbors nontemplate motifs that contribute to the specialized telomerase catalytic cycle of reiterative repeat synthesis. Most nontemplate TER motifs function through linkage with the template, but in ciliate and vertebrate telomerases, a stem-loop motif binds telomerase reverse transcriptase (TERT) and reconstitutes full activity of the minimal recombinant TERT+TER RNP, even when physically separated from the template. Here, we resolve the functional requirements for this motif of ciliate TER in physiological RNP context using the Tetrahymena thermophila p65-TER-TERT core RNP reconstituted in vitro and the holoenzyme reconstituted in vivo. Contrary to expectation based on assays of the minimal recombinant RNP, we find that none of a panel of individual loop IV nucleotide substitutions impacts the profile of telomerase product synthesis when reconstituted as physiological core RNP or holoenzyme RNP. However, loop IV nucleotide substitutions do variably reduce assembly of TERT with the p65-TER complex in vitro and reduce the accumulation and stability of telomerase RNP in endogenous holoenzyme context. Our results point to a unifying model of a conformational activation role for this TER motif in the telomerase RNP enzyme.

A Conserved Motif in Tetrahymena thermophila Telomerase Reverse Transcriptase Is Proximal to the RNA Template and Is Essential for Boundary Definition

“…Previous work has suggested multiple sites of interaction between TERT and TER (12,24). This is likely a large source of the residual binding activity in the ⌬CP2 mutant (Fig.…”

xRRM

“…Recently, we determined the solution and crystal structures of the C-terminal domain of Tetrahymena p65-TER-TERT, which contribute to different aspects of telomerase RNP assembly and function. 10,11,15,16 Some mutations in TER and TERT that abrogate or decrease activity in telomerase assembled in vitro from TERT and TER are rescued when p65 is present, which further underscores the essential role of p65 in the biogenesis and assembly of the telomerase holoenzyme. 15,17 The interaction of p65 with TER is discussed next in detail.…”

Stiffened yeast telomerase RNA supports RNP function in vitro and in vivo

“…The core consists of telomerase reverse transcriptase (TERT) and an RNA subunit (TR), which contains a short stretch of nucleotides that act as a template for TERT-catalyzed reverse transcription (Shippen-Lentz and Blackburn 1990). TR also binds other protein subunits (Lendvay et al 1996;Livengood et al 2002;Seto et al 2003), functions as a flexible scaffold for telomerase RNP assembly (Zappulla and Cech 2004), and coordinates with TERT in catalysis (Richards et al 2006;Stone et al 2007;Robart et al 2010;Berman et al 2011). While TERT is rather well conserved, the telomerase RNA is evolving extraordinarily rapidly, both in size and sequence.…”

“…Although steps have been made toward understanding how telomerase RNAs help coordinate with TERT during core enzyme activity in vitro (Richards et al 2006;Stone et al 2007;Qiao and Cech 2008;Robart et al 2010;Berman et al 2011), it is not known if telomerase RNA coordinates the essential and important accessory protein subunits for functions of the RNP holoenzyme. TLC1 may act as a simple scaffold, with no precise spatial orientation of accessory subunits necessary; i.e., the accessory protein subunits may FIGURE 1.…”